Neurogenic compositions and methods

ABSTRACT

The present invention has found that the Mts1 protein is expressed in white matter astrocytes in the spinal cord. Such expression is significantly increased following sciatic nerve injury or dorsal root injury, particularly in astrocytes surrounding dorsal funiculus containing the central processes of the injured primary sensory neurons. The present invention has further demonstrated that Mts1 proteins administered extracellularly promote neurite outgrowth from neuronal cells. Based on these surprising findings, the present invention provides compositions and methods that are useful for the treatment of various neurological conditions characterized by death, degeneration or injury of neuronal cells.

FIELD OF INVENTION

[0001] The present invention relates to the discovery of the role of theMts1/S100A4 protein in the neural system. Compositions and methods areprovided that are useful for stimulating growth of neuronal cells andtreating neuronal damage caused by disease or trauma.

BACKGROUND OF THE INVENTION

[0002] The S100 proteins comprise a large family of calcium-bindingproteins, some of which are expressed at high levels in the nervoussystem. The S100 proteins have been implicated in a wide variety offunctions, such as modulation of enzyme function, alteration ofcytoskeletal dynamics, cell adhesion and control of cell cycleprogression (Schafer et al., Trends Biochem Sci 21: 134-140, 1996).Expression of S100 protein has been shown to be associated with invasivepotential and metastatic spread of tumor cells (Inoue et al., VirchowsArch A422:351-355, 1993).

[0003] The primary structure of S100 proteins is highly conserved(Kligman et al., TIBS 13: 437-443, 1988; and Schaefer et al., TIBS 21:134-140, 1996). In solutions S100 proteins easily form dimers andcystein residues are not necessary for the noncovalent dimerization ofS100 (Mely et al., J. Neurochemistry 55: 1100-1106, 1990; Landar et al.,Biochim. Biophys. Acta 1343: 117-129, 1997; and Raftery et al., J Am.Soc. Mass Spectrom. 9: 533-539, 1988). The tertiary structure of S100proteins has been characterized (Kilby et al., Structure 4: 1041-1052,1996; Smith et al., Structure 6: 211-222, 1998; Sastry et al., Structure15: 223-231, 1998; and Matsumura et al, Structure 6: 233-241, 1998).Each S100 monomer contains two EF-hand calcium binding domains (Schaferet al., TIBS 21: 134-140, 1996). Calcium binding results in aconformational alteration and exposure of a hydrophobic patch via whichS100 proteins interact with their targets (Smith et al, Structure 6:211-222, 1998; Sastry et al, Structure 15: 223-231, 1998; Matsumura etal, Structure 6: 233-241, 1998; and Kilby et al., Protein Sci. 6:2494-2503, 1997).

[0004] Intracellular and extracellular activities of S100 proteins havealso been described (McNutt, J Cutan. Pathol. 25: 521-529, 1988).Intracellular S100 proteins interact with numerous target proteins andmodulate multiple cellular processes regulating cell growth,differentiation, metabolism and cytoskeletal structure (Zimmer et al.,Brain Res. Bulletin 37: 417-429, 1995; Schafer et al., TIBS 21: 134-140,1996; Donato, Cell Calcium 12: 713-726, 1991; and Lukanidin et al., In:Gunter U, Birchmeier W, eds. Current Topics in Microbiology andImmunology: Attempts to Understand Metastasis Formation II. Berlin,Heidelberg: Springer- Verlag 213/II, 171-195, 1996). Extracellulardisulfide-linked dimers of S100B protein have been reported to stimulateneurite outgrowth in primary cultures of cerebral cortex neurons(Kligman et al., TIBS 13: 437-443, 1988). Such activity has also beenreported for oxidized form of the recombinant S100B protein(Winningham-Major et al., J. Cell Biol. 109: 3063-3071, 1989).

[0005] The mts1/S100A4 gene, a member of the S100 gene family, wasisolated as a gene specifically expressed in metastatic murine tumorcell lines (Ebralidze et al., Genes Dev. 3: 1086-1092, 1989). Studies ofMts1-transfected non-metastatic murine cell lines and Mts1 transgenicmice both indicate that Mts1 plays an important role in tumorprogression (Grigorian et al., Gene 135: 229-238, 1993; Takenaga et al.,Oncogene 14: 331-337, 1997; Ambartsumian et al., Oncogene 13: 1621-1630,1996; and Davies et al., Oncogene 13: 1631-1637, 1996). Mts1 has alsobeen shown to affect the cytoskelton and cell motility (Takenaga et al.,Jpn. J Cancer Res. 85: 831-839, 1994) via association with stress fibers(Gibbs et al., J. Biol. Chem. 269: 18992-18999, 1994). The heavy chainof non-muscle myosin (MHC) has been identified as a target for the Mts1protein (Kriajevska et al., J. Biol. Chem. 239: 19679-19682, 1994).

[0006] The present invention identifies, for the first time, theneurogenic function of the Mts1 protein. Accordingly, the presentinvention provides novel compositions and methods useful for stimulatingneurite growth in the treatment of neural damage caused by disease orphysical trauma.

SUMMARY OF THE INVENTION

[0007] One embodiment of the present invention provides an isolatedfunctional derivative of an Mts1 protein. A preferred functionalderivative of an Mts1 protein is Mts1 del75.

[0008] Another embodiment of the present invention provides an isolatedmultimeric Mts1 protein complex. Such complex includes at least threeMts1 protein molecules or functional derivatives thereof.

[0009] In another embodiment, the present invention providespharmaceutical compositions which include an isolated functionalderivative of an Mts1 protein, or a multimeric Mts1 protein complex, anda pharmaceutically acceptable carrier. The pharmaceutical compositionscan also include one or more neurotropic factors.

[0010] In a further embodiment, the present invention provides methodsof stimulating growth of neuronal cells by administering an Mts1 proteinor a functional derivative thereof.

[0011] In a further embodiment, the present invention provides methodsof treating neurological conditions in a subject by administering to thesubject a therapeutically effective amount of an Mts1 protein or anucleotide sequence encoding an Mts1 protein. The methods of the presentinvention can be employed in the treatment of a variety of neurologicalconditions characterized by neuronal degeneration, neuronal death orinjury caused by disease, physical trauma or ischemic conditions. Suchneurological conditions include Parkinson's disease, Down's Syndrome,Alzheimer's disease, stroke, cardiac arrest, sciatic crush, spinal cordinjury, damaged sensory neurons in dorsal root ganglia and othertissues, as well as degenerative diseases of the retina.

BRIEF DESCRIPTION OF DRAWINGS

[0012]FIG. 1 depicts Mts1-immunoreactivity (IR) (A,B,E) and GFAP-IR(C,D,F) in the normal white matter of LA. (A) shows Mts1-IR in theventral and lateral funiculi, with exclusive expression in white matter.Double labeling with antibodies to Mts1 and GFAP shows that Mts1 islocalized to astrocytes (B,D) and is predominantly expressed in the cellbodies (B), while intense GFAP-IR is observed in processes as well (D).Arrowheads indicate cells that were labeled with anti-GFAP antibodies(D), but not with antibodies to Mts1 (B). (E) shows a few Mts1-positivecell bodies as well as Mts1-positive processes in paramedian septa ofthe dorsal funiculus in C3 (E), despite widespread GFAP-IR (F). Bar=200μm (A,C), 50 μm (B,D), 100 μm (E,F).

[0013]FIG. 2 depicts Mts1-IR (A) and GFAP-IR (B) in the dorsal funiculusand adjacent dorsal horn (DH) of L4 two days after unilateraltransection of dorsal roots L4 and L5. There was a marked increase inMts1-positive cell bodies and processes (A) in the white matter, and aconcomitant increased expression of GFAP (B) on the operated side(right), but no Mts1-IR in the dorsal horn (DH). Bar=200 μm.

[0014]FIG. 3 depicts Mts1-IR (A,C) and GFAP-IR (B,D) in the dorsalfuniculus of L4 one week (A,B) and two months (C,D) after unilateraltransection of L4 and L5 dorsal roots. There was a marked upregulationin the expression of Mts1 (A,C) and GFAP (B,D) on the operated side(op). The dorsal horn (DH) was completely devoid of Mts1 staining (C),despite a prominent increase in GFAP-IR (D). Bar =100 μm.

[0015]FIG. 4 depicts increased Mts1-IR (A,C) and GFAP-IR (B,D) in thegracile funiculus (A,B) and the dorsal funiculus of C3 (C,D) one weekafter ipsilateral injury to the L4 and L5 dorsal roots. Op=operatedside. Bar=100 μm.

[0016]FIG. 5 depicts double labeling with antibodies to Mts1 and GFAP(A), and double labeling with antibodies to Mts1 and themicroglia/macrophage marker ED1 (B) in the degenerating dorsal funiculustwo months after transection of the L4 and L5 dorsal roots. Mts1-IR(A,B,green) is confined to GFAP-positive astrocytes (A,red), butcompletely absent from ED1-positive cells (B,red). Bar=50 μm.

[0017]FIG. 6 depicts Mts1-IR (A,C) and GFAP-IR (B,D) in the dorsalfuniculus of L4 one week (A,B) and two months (C,D) after unilateraltransection of the sciatic nerve. There was an increased expression ofMts1 at both postoperative survival times (A,C). Mts1-IR was absent fromthe dorsal horn (C,DH). GFAP-IR was increased two months (D), but notone week (B) after injury compared to the unoperated side. Op=operatedside. Bar=100 μm.

[0018]FIG. 7A is a phase contrast micrograph of a 24 h low-densityculture of dissociated hippocampal cells of rat embryos (E18).

[0019]FIG. 7B is a phase contrast micrograph of a 24 h low-densityculture of dissociated hippocampal cells of rat embryos (E18) grown inthe presence of 5 μM recombinant Mts1/S100A4 protein.

[0020]FIG. 7C is a phase contrast micrograph of a 24 h low-densityculture of dissociated hippocampal cells of rat embryos (E18) grown inthe presence of 5 μM recombinant His-tagged 200aa C-terminal peptide ofmyosin heavy chain.

[0021]FIG. 8A depicts the dose-dependent effect of Mts1/S100A4 onneurite outgrowth in primary cultures of dissociated rat hippocampalcells. Cultures were grown in the presence of various amounts of therecombinant protein for 24 h, and neurite length per cell was measured.

[0022]FIG. 8B depicts the time-dependent effect of Mts1/S100A4 onneurite outgrowth in primary cultures of dissociated rat hippocampalcells. Hippocampal cells were seeded and allowed to attach for 1 h afterwhich recombinant Mts1/S100A4 was added to the culture (time 0). Atvarious time points afterwards, Mts1/S100A4 was removed by changingculture medium and neurite length per cell was measured 24 h afteraddition of the protein.

[0023]FIG. 8C depicts the specificity of the Mts1/S100A4 effects onneurite outgrowth in primary cultures of dissociated rat hippocampalcells. Hippocampal cells were grown for 24 h in the presence of 5 μMMts1/S100A4 and rabbit polyclonal anti-Mts1 antibodies at variousdilutions. The length of neurites in treated cultures is expressed as apercentage of the length of neurites in control cultures.

[0024]FIG. 9A depicts the effects of Mts1/S100A4, S100β, NGF and FGF onneurite outgrowth from hippocampal neurons. Cultures were grown for 24 hin the absence or in the presence of Mts1/S100A4, S100ββ, NGF or FGF atindicated concentrations. Results of a typical experiment are shown.

[0025]FIG. 9B depicts the effects of Mts1/S100A4, S100β, NGF and FGF onneurite outgrowth from PC12-E2 cells. Four individual experiments wereperformed. Results are given as mean±SEM.

[0026]FIG. 10 depicts the neurogenic effects of the wild type andmutatnt Mts1/S100A4 proteins. Hippocampal cells were grown for 24 h inthe presence of 5 μM mouse recombinant Mts1/S100A4 or in the presence of5 μM of the Mts1 mutated proteins. The length of neurites in treatedcultures is expressed as a percentage of the length of neurites incontrol cultures. Four individual experiments were performed. Resultsare given as mean± SEM.

[0027] FIGS. 11A-11C depict the profiles of the recombinant wild type(wt) Mts1 protein (11A) and two mutants, Y75F (11B) and del75 (11C) offsize exclusion chromatography (SEC). One milliliter of each protein (2mg/ml) was chromatographed on a Superdex G75 column. The column wasequilibrated with TND, eluted (1 ml/min) with the same buffer and 3-mlfractions were collected. Results of a typical experiment are shown.Relative positions of peak I, II and III are indicated with processes ofthe injured primary sensory neurons. Additionally, the present inventiondemonstrates that Mts1 proteins administered extracellularly promoteneurite outgrowth from neuronal cells.

[0028] Accordingly, the present invention employs the neurogenicactivity of the Mts1 protein and provides compositions and methods thatare useful for the treatment of various neurological conditionscharacterized by the death, degeneration or injury of neuronal cells.

[0029] By “neurogenic activity” is meant a biological activity thatinduces, stimulates, or enhances the growth, maintains the survival, orprevents the death of the neuronal cells of the central and peripheralnervous system of a mammal. The activity can manifest as differentiationof neurons, extension of neuritic processes (i.e., outgrowth orelongation of neurites), or innervation of neuritic processes into atissue.

[0030] One embodiment of the present invention provides an isolatedfunctional derivative of an Mts1 protein.

[0031] “An Mts1 protein” as used herein, refers to a wild type Mts1protein of a mammalian origin, such as human, murine and the like.Preferred Mts1 proteins of the present invention include human Mts1 (SEQID NO: 1) and murine Mts1 (SEQ ID NO: 2), which are also described inU.S. Pat. No. 5,801,142 and Ebralidze et al., Genes Dev. 3: 1086-1092,1989, respectively.

[0032] “A functional derivative of an Mts1 protein” refers to a modifiedMts1 protein having one or more amino acid substitutions, deletions orinsertions, which retains substantially the neurogenic activity of awild type Mts1 protein. By “substantially” is meant at least about 35%,preferably, at least about 40%.

[0033] In accordance with the present invention, a preferred functionalderivative of a wild type Mts1 protein is Mts1-del75, i.e., deletion ofthe Tyr residue at the position 75 in human or murine Mts1 protein, orthe corresponding Tyr in any other mammalian Mts1 proteins. It has beendetermined by the present inventor that Mts1-del75 is able to formpolymers and confers about 70% neurogenic activity compared to a wildtype Mts1 protein. Another Mts1 mutant which has all four Cysteineresidues mutated to Serine (designated herein as “4S”) retains about 40%of the neurogenic activity of a wild type Mts1 protein.

[0034] Those skilled in the art can use any of the well-known molecularcloning techniques to generate Mts1 derivatives having one or more aminoacid substitutions, deletions or insertions. See, for example, CurrentProtocols in Molecular Cloning (Ausubel et al., John Wiley & Sons, NewYork). Once a modified Mts1 protein is made, such protein can be testedin functional assays to determine whether such modified protein exhibitsneurogenic activity.

[0035] In accordance with the present invention, the neurogenic activityof an Mts1 protein or protein complex can be determined by a number ofassays. A typical functional assay is described in Example 2hereinbelow. Briefly, an Mts1 protein is added in various doses in theculture medium of neuronal cells, such as hippocampal neuronal cells, orPC-12 cells. The cells can be kept exposed to the protein for a certainperiod of time and the outgrowth of neurites from the cultured cells aremonitored. Parameters such as the length of the longest neuriteextension, the number of neurite branches per cell, and the totalneurite length per cell, are measured. The determination as to whether amodified Mts1 protein possesses neurogenic activity can be made bycomparing these parameters with those values of a wild type Mts1 proteinand those values of a control protein without neurogenic activity. Otherassays which can be employed for such determination include, e.g., thestandard assay of endothelial cell motility in Boyden Chamber.

[0036] Another embodiment of the present invention provides an isolatedmultimeric Mts1 protein complex.

[0037] In accordance with the present invention, it has been found thatthe neurogenic activity of Mts1 is associated with the polymeric formscomposed of three or more Mts1 protein molecules. Not intending to bebound by any theory, it is proposed herein that the Mts1 proteinmediates its neurogenic effects via a cell surface receptor whichrecognizes polymeric forms of the Mts1 protein.

[0038] According to the present invention, the terms “a multimeric Mts1protein complex” and “a polymeric Mts1 protein complex” as used hereinrefer to a complex having at least three, i.e., three or more, moleculesof an Mts1 protein or a functional derivative of an Mts1 protein. Thecomplex can have a Mw of at least about 30 kd, more preferably, at leastabout 100 kd, and up to about 200 kd, as determined by, e.g.,size-exclusion chromatography.

[0039] In accordance with the present invention, the Mts1 proteinmolecules in the complex can be held together by covalent and/ornon-covalent interactions among Mts1 protein molecules. For example,there are four Cys residues in both human and murine Mts1, which canform intramolecular disulfide bonds under appropriate conditions therebyleading to formation of polymeric Mts1 complexes. The present inventionalso contemplates polymeric Mts1 complexes formed by chemicalcross-linking reagents. Chemical cross-linking reagents and use thereofin making multimeric protein complexes are well known in the art. Inaccordance with the present invention, a Mts1 protein complex havingneurogenic activity can be formed through non-covalent interactionsamong Mts1 molecules as well. For example, the present inventionprovides that Mts1-4S, while unable to form any intramolecular orintermolecular disulfide bonds, is able to form polymers and confersneurogenic activity at a level of about 40% of that of a wild type Mts1protein.

[0040] The Mts1 complexes of the present invention can be isolated by avariety of methods. For example, an Mts1 protein can be dissolved insolution under conditions that favor the formation of polymers, e.g., asaline solution of about 0.15 M NaCl, pH7.5 with a Mts1 concentrationhigher than, preferably, 1 mg/ml. Afterwards, the solution can besubjected to an appropriate chromatography procedure using, e.g.,Size-Exclusion-Column euqilibrated with a TND buffer (50 mM Tris-HCl,150 mM NaCl, 1 mM DTT, pH 7.5). The Mts1 protein can be eluted using thesame TND buffer, and fractions containing polymers can be collected andseparated from the fractions containing dimers. Such procedure isdescribed in Example 3 hereinbelow. An Mts1 protein can also besubjected to chemical cross-linking prior to chromatography or fractionprocedures. Those skilled in the art can make modifications whenappropriate and necessary.

[0041] In another embodiment, the present invention providespharmaceutical compositions which include a functional derivative of anMts1 protein, or an isolated multimeric Mts1 protein complex composed ofat least three Mts1 protein molecules.

[0042] The pharmaceutical compositions of the present invention can beemployed to promote neuronal cell growth or maintain the survival ofneuronal cells in the treatment of neurological conditions characterizedby the death, degeneration or injury of neuronal cells.

[0043] The functional derivative or the protein complex of an Mts1protein for use in the pharmaceutical compositions can be modifiedaccording to procedures known in the art in order to enhance penetrationof the blood-brain barrier. For example, U.S. Pat. No. 5,604,198discloses that a molecule can be conjugated to a hydrophobic carrierwhich enhances the permeability of the blood brain barrier (BBB). WO90/14838 teaches chemical modifications of a protein by increasinglipophilicity, altering glycosylation or increasing the net positivecharge in order to enhance the BBB permeability of the protein.

[0044] According to the present invention, the pharmaceuticalcompositions can also include one or more neurotropic factors.

[0045] Neurotropic factors are proteins which promote the survival ofneurons, some of which are also capable of promoting neurite outgrowthand glial cell restoration or inducing cells to secrete otherneurotropic factors. Preferred neurotropic factors for use in thepresent pharmaceutical compositions are those to which a broad range ofcell types respond. Examples of preferred neurotropic factors includemembers of the BDNF/NGF family, such as bFGF (basic fibroblast growthfactor), aFGF (acidic fibroblast growth factor), CNTF (ciliaryneurotrophic factor), NGF (nerve growth factor), BDNF (brain-derivedneurotrophic factor), GDNF (glial cell line-derived neurotrophicfactor), NT-3 (neurotrophin-3), NT-4/5 (neurotrophin 4/5), IGF-1(insulin growth factor-I), IGF-II (insulin growth factor-II), andfunctional peptide fragments thereof. Human neurotropic factors andfunctional derivatives are preferred.

[0046] The active ingredients of the pharmaceutical compositions arepreferably provided in a pharmaceutically acceptable carrier. Thecarrier can be liquid, semi-solid, e.g. pastes, or solid carriers.Except insofar as any conventional media, agent, diluent or carrier isdetrimental to the recipient or to the therapeutic effectiveness of theactive ingredients contained therein, its use in the pharmaceuticalcompositions of the present invention is appropriate. Examples ofcarriers include oils, water, saline solutions, gel, lipids, liposomes,resins, porous matrices, binders, fillers and the like, or combinationsthereof. The carrier can also be a controlled release matrix whichallows a slow release of the active ingredients mixed or admixedtherein. Examples of such controlled release matrix material include,but are not limited to, sustained release biodegradable formulationsdescribed in U.S. Pat. Nos. 4,849,141 to Fujioka et al., 4,774,091 toYamashira, 4,703,108 to Silver et al., and Brem et al.(J. Neurosurg. 74:441-446, 1991), all of which are incorporated herein by reference.

[0047] In accordance with the present invention, a Mts1 functionalderivative or an Mts1 polymeric complex can be combined with the carrierin solutions or in solid phase, preferably in a manner that favors thestablization of the polymeric conformation of the Mts1 protein. If themixing step is to be performed in liquid phase, Mts1 proteins can bedissolved together with a carrier in solutions such as saline (about0.15M NaCl pH 7.5) with an Mts1 concentration of higher than,preferably, 1 mg/ml. If the mixing is to be performed in solid phase,the Mts1 polymeric proteins can be freeze-dried first to preserve thepolymeric conformation, then admixed with the carrier. The mixture canbe made in formulations suitable for injections, implantations,inhalations, ingestions and the like.

[0048] In a further embodiment, the present invention provides methodsof stimulating growth of neuronal cells by administering an Mts1protein, a functional derivative of an Mts1 protein, or a proteincomplex thereof, to such neuronal cells.

[0049] According to the present invention, an Mts1 protein or afunctional derivative or complex thereof, can be administered toneuronal cells that are cultured in vitro. This aspect of the inventionis particularly useful in regeneration of neurons forautotransplantation or neuron replacement as an alternative treatmentprocedure to brains of patients with neurological disorders. Techniquesof culturing neurons in vitro fare known in the art and are describedin, e.g., U.S. Pat. Nos. 5,483,892, 5,753,506, 5,898,066, and 5,667,978,Mou et al. J. Comp. Neurol. 386: 529 (1997), and Tan et al. CellTransplant 5: 577 (1996), the teachings of which are incorporated hereinby reference.

[0050] In a further embodiment, the present invention provides methodsof treating neurological conditions in a subject by administering to thesubject a therapeutically effective amount of an Mts1 protein, afunctional derivative thereof, or a nucleotide sequence encoding an Mts1protein.

[0051] The methods of the present invention can be employed in thetreatment of a variety of neurological conditions characterized byneuronal degeneration, neuronal death or injury caused by disease,physical trauma or ischemic conditions. Such neurological conditionsinclude Parkinson's disease, Alzheimer's disease, Down's Syndrome,stroke, cardiac arrest, sciatic crush, spinal cord injury, multiplesclerosis, peripheral neuropathies associated with diabetes, motorneurondiseases, damaged sensory neurons in dorsal root ganglia and othertissues, as well as degenerative diseases of the retina.

[0052] By “treating” is meant prevent or inhibit neuronal degenerationor neuronal death, promoting or stimulating neuronal growth such thatthe symptoms of the disease condition are prevented or alleviated.

[0053] In accordance with the methods of the present invention, an Mts1protein can be first treated to enrich the polymeric forms, or can beused directly, as certain percentage of the molecules spontaneouslyassociate with each other to form polymers in solution. An Mts1 proteinor a functional derivative thereof can be modified in order to enhancepenetration of the blood-brain barrier as described hereinabove.

[0054] Nucleic acid sequences encoding an Mts1 protein can also beemployed in the methods of the present invention. Such sequences arepreferably provided in an expression vector. Expression vectors for usein the present methods include any appropriate gene therapy vectors,such as nonviral (e.g., plasmid vectors), retroviral, adenoviral, herpessimplex viral, adeno-associated viral, polio viruses and vacciniavectors. Examples of retroviral vectors include, but are not limited to,Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV)-derived recombinant vectors. Multiple teachings of gene therapyare available to those skilled in the art, e.g., W. F. Anderson (1984)“Prospects for Human Gene Therapy” Science 226: 401-409; S. H. Hughes(1988) “Introduction” Current Communications in Molecular Biology 71:1-12; T. Friedman (1989) “Progress Toward Human Gene Therapy” Science244: 1275-1281 and W. F. Anderson (1992) “Human Gene Therapy” Science256: 608-613. Preferred vectors include neurotropic vectors such asherpes simplex viral vectors (U.S. Pat. No. 5,673,344 to Kelly et al.and adenoviral vectors (Barkats et al., Prog. Neurobiol. 55: 333-341,1998).

[0055] Mts proteins or Mts1-encoding nucleic acid molecules can be usedalone or in conjunction with one or more neurotropic factors describedhereinabove, including members of the BDNF/NGF family such as bFGF,aFGF, CNTF, NGF, BDNF, GDNF, NT3, NT4/5, IGF-1 and IGF-II, as well asthe functional peptide fragments identified thereof. Human neurotropicfactors are preferred for treating a human subject.

[0056] The therapeutically active ingredients, i.e., Mts1 proteins ornucleic acid molecules, alone or in conjunction with neurotropicfactors, can be combined with a pharmaceutically acceptable carrier andprepared in formulations suitable for injections, implantations,inhalations, ingestions and the like. Pharmaceutically acceptablecarriers are described hereinabove and include oils, water, salinesolutions, gel, lipids, liposomes, resins, porous matrices, binders,fillers and the like, or combinations thereof.

[0057] According to the present invention, these therapeuticcompositions can be administered to the subject being treated bystandard routes, including the oral, ophthalmic nasal, topical,transdermal, parenteral (e.g., intravenous, intraperitoneal,intradermal, subcutaneous or intramuscular), intracranial,intracerebral, intraspinal, intravaginal, intrauterine, or rectal route.Depending on the condition being treated, one route may be preferredover others, which can be determined by those skilled in the art. Forexample, topical route can be chosen when the target area includestissues or organs readily accessible by topical application, such asneurological conditions of the eye or the facial tissue. For certainconditions, direct injection or surgical implantation in the proximityof the damaged tissues or cells may be preferred in order to avoid theproblems presented by BBB. Successful delivery to CNS (Central NervousSystem) by direct injection or implantation has been documented. See,e.g., Otto et al., J. Neurosci. Res. 22: 83-91 (1989); Goodman &Gilman's The Pharmacological Basis of Therapeutics, 6^(th) ed, pp 244;Williams et al., Proc. Natl. Acad. Sci. USA 83: 9231-9235 (1986); andOritz et al., Soc. Neurosci. Abs. 386: 18 (1990).

[0058] According to the present invention, the therapeutic ingredientsare preferably administered to the subject in need thereof as early aspossible after the neuronal injury or death occurs in order to achievethe best therapeutic efficacy.

[0059] The amount of an Mts1 protein, a functional derivative, or anMts1-encoding nucleic acid molecule to be therapeutically effectivedepends on the disease state or condition being treated and otherclinical factors, such as weight and physical condition of the subject,the subject's response to the therapy, the type of formulations and theroute of administration. The precise dosage to be therapeuticallyeffective and non-detrimental to the subject can be determined by thoseskilled in the art. As a general rule, the therapeutically effectiveamount of Mts1 protein can be in the range of about 0.01 mg to about 10mg per kilogram of body weight; preferably, in the range of about 0.1 mgto about 5 mg per kilogram of body weight. The therapeutically effectivedosage of an Mts1 protein can be in the range of about 0.5 μg to about 2mg per unit dosage form. A unit dosage form refers to physicallydiscrete units suited as unitary dosages for mammalian treatment: eachunit containing a pre determined quantity of the active materialcalculated to produce the desired therapeutic effect in association withany required pharmaceutical carrier. The methods of the presentinvention contemplate single as well as multiple administrations, giveneither simultaneously or over an extended period of time.

[0060] This invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof. The terms and expressions which have been employed inthe present disclosure are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof. It is to be understood that variousmodifications are possible within the scope of the invention. All thepublications mentioned in the present disclosure are incorporated hereinby reference.

EXAMPLE 1 MtS1 Expression Is Up-Regulated After Peripheral or DorsalRoot Injury

[0061] Introduction of the Experimental Model

[0062] The primary sensory neurons of the spinal cord with their cellbodies located peripherally, send out dichotomizing processes, onebranch projecting peripherally to innervate peripheral tissues andorgans, the other branch entering the CNS via spinal dorsal roots.Dorsal root axons terminate in a specific pattern in the gray matter ofthe dorsal horn. In addition, collaterals of myelinated primary sensoryaxons ascend in the dorsal funiculus of the white matter to the lowerbrainstem where they terminate in the dorsal column nuclei.

[0063] Injury to the dorsal root (rhizotomy) and injury to theperipheral branches produce markedly different morphological andmolecular changes in the affected neurons. However, both injuries areassociated with prominent responses in surrounding non-neuronal cells inthe CNS, particularly astrocytes and microglia/macrophages. Injury tothe peripheral branches, e.g. by section of the sciatic nerve, inducesdegenerative as well as growth-associated changes (transganglionicchanges) in the central terminals and axons of the injured neurons(Aldskogius et al., Oxford Univ Press. pp 363-383, 1992; Woolf et al.,Neurosci 34: 465-4678, 1990; and Woolf et al., J Comp Neurol 360:121-134, 1995). Concomitantly, microglial cells proliferate (Gehrmann etal., Restor Neurol Neurosci 2: 181-198, 1991; Eriksson et al., Exp BrainRes 114: 393-404, 1993; and Persson et al., Primary Sensory Neuron 1:47-64, 1995), and express various inflammatory mediators (Liu et al.,Neurosci 68: 167-179, 1995), while astrocytes upregulate the expressionof their major intermediate filament, glial fibrillary acidic protein(GFAP) (Gilmore et al., Glia 3:342-349, 1990) but do not proliferate.Injury to the central primary sensory process by section of the dorsalroot, results in complete disintegration (Wallerian degeneration) of thesegment of the axon no longer in continuity with the parent cell body.The non-neuronal response to this degeneration includes proliferation ofmicroglia, that gradually develops into macrophages, as well asproliferation of astrocytcs and a rapid increase in the expression ofGFAP in astrocytes (Liu et al., Glia 23:221-238, 1998).

[0064] Materials and Methods

[0065] Thirty-two adult, female, Sprague-Dawley rats (160-180 g bodyweight) were used for the study. Prior to surgery and perfusion, animalswere anaesthetized with chloral hydrate (35 mg/kg body weight i.p.).

[0066] Twelve animals were subjected to section of the left sciaticnerve at midthigh level. Two animals (n= 2) were analyzed at eachpostoperative survival time (1 day, 2 days, 3 days, 7 days, 1 month and2 months). In 18 animals, the left lumbar dorsal roots L4 and L5 wereexposed via a partial laminectomy and sectioned close to thecorresponding dorsal root ganglia (n=3 for each postoperative survivaltime). At the indicated postoperative survival time, the animals wereperfused via the left ventricle first with saline (37° C.) followed by asolution of 4% formaldehyde (w/v) and 14% saturated picric acid (v/v) ina 0.15M phosphate buffer (pH 7.4, 4° C.). Two intact control animalswere perfused in the same way. The L4-LS and C3 spinal cord segments aswell as the brainstem were removed, postfixed for about one and halfhours, and subsequently stored overnight in refrigerator. Serial, 14 μmtransverse sections were cut on a cryostat and processed forimmunofluorescence. In addition, sets of sections were cut at 5 μm toprovide material for optimal microphotography.

[0067] Sections were briefly air-dried and washed in phosphate bufferfor 5-10 mins prior to incubation in BSA and 0.3% Triton X100 (Sigma,USA) for one hour at room temperature. Sections were incubated overnightat 4° C. with antibodies against Mts1 (rabbit polyclonal, 1:1000). Theimmune complex was visualized with FITC-conjugated sheep anti-rabbit IgG(Jackson, 1:40). For double labeling experiments, anti-Mts1 antibodieswere combined with one of the following antibodies: (1) anti-GFAP(astrocytes, mouse monoclonal (Serotec, U.K.), 1:3), (2) OX42(microglia, mouse monoclonal (Serotec, U.K.), 1:600), or (3) ED1(phagocytic microglia/macrophages, mouse monoclonal (Serotec, U.K.),1:400). The cell marker antibodies were visualized with rhodamine(TRITC)-conjugated anti-mouse IgG. Sections were viewed and photographedin a Nikon Eclipse fluorescence microscope equipped with filter forsimultaneous examination of FITC and TRITC fluorescence.

[0068] Intact Control Animals

[0069] Mts1 immunoreactivity (IR) was observed in the white matter ofthe L4 and C3 segments of the spinal cord as well as in the brainstem.The most prominent staining appeared in the ventral and lateral funiculias processes radiating from the subpial region and towards the graymatter, leaving, however, its immediate white matter surroundings freefrom Mts1-IR (FIG. 1, A and B). Mts1-IR cell bodies were typicallylocated in the subpial region as well as about midway between thisregion and the gray matter. Double labeling with glial cell markersshowed colocalization between Mts1 and anti-GFAP (FIG. 1, B and D), buta minority of GFAP-positive cells was not labeled with Mts1. However,astrocytes which did express Mts1, showed a more complete labeling oftheir cell bodies with anti-Mts1 than with anti-GFAP. Conversely,GFAP-IR processes were usually only partially labeled with anti-Mts1(FIG. 1, B and D).

[0070] The levels Of Mts1-IR were considerably lower in the dorsalfuniculus of L4-L5 and C3 as well as in the dorsal white matter of thebrainstem compared to the ventral and lateral funiculi. Only someMts1-positive profiles were observed (FIG. 1, E); there was no apparentdifference in GFAP staining (FIG. 1, F).

[0071] Dorsal Root Injury

[0072] Since the uninjured and injured sides of the spinal cord werenext to each other, changes in Mts1-IR as a result of sciatic nerve ordorsal root transaction could be unambiguously identified. The firstsign of an upregulation of Mts1-IR in the L4 dorsal funiculus wasobserved two days after dorsal rhizotomy (FIG. 2, A). This wasparalleled by an increased staining for GFAP in the same area (FIG. 2,B). At this state, large Mts1-positive cells appeared in the areaoccupied by the injured primary sensory axons in the dorsal funiculus.The difference between the degenerating zone in the dorsal funiculus andthe uninjured white matter gradually became stronger with increasingpostoperative survival time (FIG. 3, A and B), and was very intense attwo months after injury (FIG. 3, C and D). Importantly, the gray matter,including the dorsal horn termination area of the injured primaryafferents, was always Mts1 negative, despite a marked up-regulation ofGFAP-IR in the termination sites of the injured primary afferent fibers(FIG. 3, B and D).

[0073] Increased immunoreactivity for Mts1 and GFAP also appeared alongthe central processes of the injured lumbar primary sensory afferents inthe dorsal column of C3 and in the gracile nucleus. At one week afterrhizotomy Mts1-IR was up-regulated concomitantly with GFAP-IR in thegracile funiculus and nucleus in the lower brainstem (FIG. 4, A and B)and in C3 in the circumscribed area of the dorsal funiculus containingthe degenerating ascending primary sensory afferents (FIG. 4, C and D).

[0074] Double labeling with markers for Mts1 and for astrocytes (GFAP)or for microglia/macrophages (antibodies OX42 or ED1), showed overlapbetween Mts1-IR and GFAP-IR in the dorsal funiculus (FIG. 5, A), butnone between Mts1- and OX42 or EDI-IR (FIG. 5, B).

[0075] Sciatic Nerve Injury

[0076] Mts1-IR in the ipsilateral dorsal funiculus was upregulated firstat one week after sciatic nerve injury (FIG. 6, A) and showed agradually increasing expression with longer survival times. However, atthis postoperative time there was no increase in GFAP-IR (FIG. 6, B) inthe dorsal funiculus, although there was an upregulation in the dorsalhorn. The extent of Mts1-IR was never as great after sciatic nerveinjury as after dorsal root lesions, even at the longest postoperativesurvival time of two months, when it coincided with an increased GRAP-IR(FIG. 6, C and D). The upregulation of Mts1 was always confined to thesomatotopically appropriate area for sciatic nerve afferents in thedorsal funiculus, and did not include its most dorsomedial part,occupied by uninjured ascending sacral primary afferents, nor itsventralmost part occupied by the corticospinal tract. The gray matterwas always free from Mts1-IR, despite an upregulation of GFAP-IR (FIG.6, C and D). Double labeling with antibodies to Mts1 and with glial cellmarkers showed colocalization only with antibodies to GFAP (cf. FIG. 6,C and D).

EXAMPLE 2 Recombinant Mts1 Protein Stimulates Neurite Outgrowth in vitro

[0077] Murine Mts1 protein sequence was described by (Ebralidze et al.,Genes Dev. 3, 1086-1092, 1989). cDNA fragments encoding the murine Mts1protein and mutant Mts1 proteins containing a single mutation Y75F, atyrosine deletion (del75) or cysteine/serine substitutions (4S) werecloned into pQE30 expression vector (QIAGEN, Inc., CA) and partiallysequenced. Expression of recombinant His₆-tagged proteins was induced byisopropy-1-thio-β-D-galactopyranoside, and bacterial lysates were usedfor isolation of proteins according to the the manufacturer's protocol.Proteins were separated on SDS-PAGE, followed by Western blot analysisas described by Kriajevska et al. (J. Biol. Chem. 273: 9852-9856, 1998).

[0078] Hippocampus was isolated from Wistar rat embryos at gestationalday 18 and dissociated cells were obtained as descried by Maar et al.(J. Neurosci. Res. 47: 163-172, 1997). Briefly, hippocampal tissue washomogenized, trypsinized and washed in the presence of DNAse I andtrypsin inhibitor. Hippocampal cells were seeded in 8-well LabTekcoverslides at a density of 5×10³ cells/CM², maintained in neurobasalmedium supplemented with B27 supplement, 4 mg/ml bovine serum albumin(BSA), penicillin (100 U/ml) and streptomycin (100 μg/ml). Cells weregrown for 24 h in a humidified atmosphere with 5% CO₂.

[0079] The neurogenic effect of Mts1 was analyzed-by computer-assistedmorphometry. The embryonic hippocampal neurons of 18-day rats werecultured with and without the Mts1 protein at low cell density in serumfree defined medium. Cells were then fixed in 4% paraformaldehyde andstained for 20 min in Commassie blue R250 (4 g/l in 45% v/v ethanol and45% v/v acetic acid). Coverslides were observed in a Nikon Diaphot 300inverted microscope using phase contrast optics (Nikon Plan 20x). Videorecording was made with a CCD video camera (Burle, USA). 512×512 pixelimages were stored in a computer using the PRIGRA software package(Protein Laboratory, University of Copenhagen). To measure neuriteoutgrowth from hippocampal neurons a simple procedure developed at theProtein Laboratory and based on stereological principles was used.Briefly, by means of the software package “ProcessLenghth” (ProteinLaboratory, University of Copenhagen), an unbiased counting framecontaining a grid with a certain number of test-lines was superimposedon images of the cell cultures. The number of intersections of cellularprocesses with the test-lines was counted and related to the number ofcell bodies, thereby allowing qualification of the total neurite lengthper cell by means of the equation, L=π/2×d×J, in which L is the neuriticlength in micrometers, d is the vertical distance between two test linesand J is the number of intersections between the test lines and theneurites.

[0080] It was observed that hippocampal neurons cultured without theMts1 protein did not differentiate by extending processes (FIG. 7A).Treatment of hippocampal neurons with the recombinant His-tagged wt Mts1protein of 5 μM for 12 hours had a robust effect on theirdifferentiation (FIG. 7B). Neurons extended multiple, long branchingprocesses. Cell cultures treated with the recombinant His-tagged 200aaC-terminal peptide of the myosin heavy chain (Kriajevsta et al., J.Biol. Chem 273: 9852-56, 1998) for 24 h, revealed minimal morphologicalchanges in comparison to control cultures (FIG. 7C).

[0081] The stimulation of neurites outgrowth by the recombinant Mts1protein was time- and dose-dependent. Mts1 was effective in themicromolar concentration range, with the maximal growth-stimulatoryactivity being 5-10 μM (FIG. 8A). Mts1 treatment increased the totallength of neurite per cell when compared to the control, as well as thenumber of neurites (7 fold), the length of the longest neurite (14 fold)and the number of branches (25 fold) per cell (Table 1). TABLE 1 NeuriteInduction in Hippocampal Neurons in Vitro Following Treatment with theRecombinant Mouse Mts1/S100A4 Protein Total neurite Length of the lengthper longest neurite Neurite Neurites cell per cell branches per cell(μM) (μM) per cell Control 0.29 ± 0.06 12.6 ± 1.3  3.43 ± 0.5 0.013 ±0.01 Mts1 (5 μM) 2.12 ± 0.3  93 ± 17 49.5 ± 1.5  0.36 ± 0.08

[0082] The duration of the Mts1 protein treatment required forhippocampal cells to extend neurites was also determinaed. In theseexperiments, Mts1 was added at the time (time 0) when seeded cells wereallowed to attach for 1 h. At various time points Mts1 was removed bychanging culture medium, and neurite outgrowth was measured 24 h later.Cells exposed to the Mts1 protein for 15-30 min already displayed a4-fold increase in the total length of neurites when compared to controlcells. The response of cells exposed to Mts1 for more that 1.5 h wasobvious and indistinguishable after further incubation for 4, 6, 16 or24 h, respectively (FIG. 8B). These data indicate that continuousexposure of cells to Mts1 for 24 h is not required and that there is anearly period, approximately 1-1.5 hour, when the presence of Mts1 isessential for the maximal neurite outgrowth.

[0083] The specificity of Mts1 neurogenic activity was tested byexamining the activity of the Mts1 protein after incubation withantibodies to Mts1. The Mts1 protein was mixed with serial dilutions ofpolyclonal anti-Mts1 antibodies in growth medium, incubated for 1 h andapplied to hippocampal cells. FIG. 8C shows that incubation of Mts1 withantibodies directed against Mts1 reduced the neurite extension in areverse proportion to the antibodies dilutions. Incubation of Mts1 withcontrol IgG, anti nonmuscle myosin or normal rabbit serum, did notreduce the response.

[0084] The neurogenic activity of Mts1 was compared with the activitiesof other neurotrophic growth factors, including FGF (Fibroblast GrowthFactor), NGF (Nerve Growth Factor) and members of S100 Ca²+-bindingprotein— S100α and S100β. Neurite outgrowth from hippocampal neurons wasnot stimulated by FGF, NGF or S100β (FIG. 9A). Treatment with S100α didnot affect hippocampal cultures either. Moreover, NGF actually inhibitedneurite outgrowth at high concentrations (5-10 μM).

[0085] To assess the possibility that lack of responsiveness ofhippocampal cells to FGF, NGF, and S100β reflected cell specificactivity of these neurotropic factors, PC-12 cells were tested. As shownin FIG. 9B, Mts1 and S100β showed equal neurite outgrowth stimulatoryactivity in the PC-12 cells. As shown in FIG. 9B, Mts1 and S100β showedequal neurite outgrowth stimulatory activity in the PC-12 cell systemthat was twice as high compared with that in the hippocampal cells. Incontrast, neurite extension effect of FGF and NGF on cultured PC-12cells was significantly higher that on hippocampal cells. The dataindicate that the stimulatory effects of different neurotrophic factorsare cell specific, and Mts1 is a potent activator of neurites outgrowthof hippocampal cells.

EXAMPLE 3 Structural Requirements for the Mts1 Neurite OutgrowthPromoting Activity

[0086] To determine the structural elements in the Mts1 protein that arerequired for promoting neurite outgrowth, three Mts1 mutatant proteinswere tested. In one of the mutants, Tyrosine75 was substituted toPhenylalanine (Y75F). In the other mutant Tyrosine75 was deleted(del75). It was found that Del75 could not form dimers in the yeasttwo-hybrid system, while the Y75F mutant formed perfect dimers in theyeast with an efficiency even higher than wt Mts1.

[0087] When these two mutant Mts1 proteins were tested in the in vitrosystem of cultured hippocampal cells, it was found that Y75F did notstimulate neurite outgrowth from hippocampal cells. In contrast, cellsincubated with del75 for 24 h displayed abundant neurites, although thedegree of neurite outgrowth was generally lower than that obtained withthe wild type Mts1 (FIG. 10).

[0088] To examine whether disulfide bonds contribute to the neurogenicactivity of the Mts1 protein, the Mts1 mutant termed 4S was used, inwhich all four cysteins (at positions 76, 81, 86 and 93) of the Mts1protein were changed to serines. It was found that 4S was able to formdimers in the yeast two hybrid system, but unable to interact with theheavy chain of myosin in a gel overlay assay. When tested for theability to stimulate neurite outgrowth, the 4S mutant showed 40% of theneurogenic activity of that of wt Mts1 (FIG. 10).

[0089] In order to determine which conformational forms of Mts1 wereactive with regard to neurogenic activity, size-exclusion chromatography(SEC) of the recombinant Mts1 and the Mts1 mutants were performed. ASuperdex75 column (1.5 cm²×90.0 cm) was equilibrated with a TND buffer(50 mM Tris-HCI, 150 mM NaCl, 1 mM DTT, pH 7.5) with and without 5 mMCaCl₂. The column was calibrated for molecular weight determinationsusing gel filtration chromatography standard (Bio-Rad). The standardproteins included Vitamin B-12 (MW 1.35 kDa), equine myoglobin (MW 17.0kDa), chicken ovalbumin (MW 44.0 kDa), bovine gamma globulin (MW 158.0kDa), thyroglobulin (MW 670.0 kDa). 1 ml of the mixed proteins standard(2 mg/ml) was loaded onto the column and 3 ml fractions were collectedand monitored with A₂₈₀ readings. Dextran blue was applied to the columnto determine its void volume. The K_(av) values were determined for eachprotein and plotted versus the log of the molecular weight of thestandard K_(av)=(V_(e)−V₀)/(V_(t)−V_(o)) (V_(e) is the elution volume atthe peak apex, V_(o) is the void volume, and V_(t) is the total columnvolume; see Landar et al., Biochim. Biophys. Acta 1343: 117-129, 1997).

[0090] 1 ml samples of the Mts1 protein or mutants were applied onto thecolumn and a K_(av) value was determined in each case. The molecularweight of Mts1 was determined by comparing its K_(av) value to thosefound for the standard proteins. Gel filtration chromatographyexperiments were performed under different conditions: presence ofreducing agent, 2 mM calcium or 2 mM EDTA, 0.5M or 0.15M NaCl. Thefractions were assayed by both SDS-PAGE and the neurite outgrowth test.Under either condition, the eluted material showed a broad profile ofdistribution with molecular masses ranging approximately from 30 to 200kDa. (FIG. 11A). Approximately half of the recombinant wild type Mts1protein was eluted as a high molecular weight complex. The distinct peakof a dimer was consistently detected among different batches of freshlyprepared recombinant Mts1, whereas the elution profile of a highermolecular mass material was less reproducible and varied in differentMts1 preparations.

[0091] The elution profile of the Y75F mutant was different as shown inFIG. 11B. 85% of the Y75F protein was eluted from gel filtration columnsas a single peak with a molecular weight of a dimer, and 15% asmaterials of higher molecular weights ranging from 30 to 100 kDa (FIG.11B). The elution profile of the mutant del75 was different from eitherthe wild type Mts1 or the Y75F mutant protein. Major part of the del75protein was eluted as materials of high molecular weights rangingapproximately from 40 kDa to 200 kDa.

[0092] It was further found that the elution profiles of all proteinswere not influenced by alterations in the Ca++ concentration, nor bychanges from reducing to non-reducing conditions, nor by changes inionic strength.

[0093] Different fractions eluted from the column, named peaks I, II andIII for all three tested proteins, were analyzed for the presence ofMts1 by Coomassie staining and Western blot analysis (FIG. 11D). TheMts1 protein under reducing condition yielded one 11 kDa band in allanalyzed fractions. Western blot analysis with affinity purifiedantiserum confirmed the Mts1 origin of the bands described as monomer.SDS gel patterns of the two mutant proteins were similar to Mts1.

[0094] The relative contribution to the neurogenic activity of differentforms of the Mts1 protein, eluted from the SEC column as peaks I, II andIII, was tested. Inserts in FIGS. 11A-11C show that high molecularweight complexes (100-200 kDa ) of wt Mts1 as well as del75 mutantstimulated neurite outgrowth. Peak I demonstrated the highest activity.The neurogenic activity of the protein in peak II was less reproducibleand accounted for 30% of the activity observed in the peak I. Dimericforms (peak III) of wt Mts1 and two Mts1 mutants (Y75F and 4S) showed noactivity at any dose tested. The data indicate that the ability tostimulate neurite extension is attributed to the polymeric fraction ofthe Mts1 molecules with unidentified structural conformations.

[0095] In order to monitor the polymerization of Mts1 and to determinethe molecular weight of the polymers more precisely, the recombinantMts1 protein was analyzed by Dynamic Light Scattering, a standardtechnique for determination of the molecular weight of globular proteins(Berne et al., Dynamic Light Scattering, Chap. 5, Wiley, New York,1976). The diffusion coefficient (D_(t)) and calculated molecular weightwere determined with DLS using Dyna Pro 801 Molecular Sizing Instrument(Protein Solutions Inc.). All readings were recorded at 18° C. Allsamples were filtrated through a 0.02 μm membrane (Whatman) beforemeasurements. Protein solutions were injected into a 25 μl cell(cuvette) and illuminated by a 25W 750 nm wave length laser. Data werefitted with the Dynamics Version 4.0 software package. The molecularweight (M.W.) was calculated by two alternative models. According to thefirst model, M.W. was estimated from the hydrodynamic radius (R_(h))using an empirically derived relationship between the R_(h), and M.W.values for a number of well-characterized globular proteins in abuffered aqueous solution, assuming that the protein holds a standardglobular shape and density. In the second model, thevolume-shape-hydration relationship was used, in which model thecalculation required the values of the hydrodynamic size, partialspecific volume, and frictional ratio. (The value of partial specificvolume (V) is 0.707 in the absence of Ca²⁺ and V increases when Ca²⁺ isadded (Mani et al., FEBS Lett. 166, 258-262, 1984). The value offrictional ratio (f) is 1.45 and f decreases when Ca²⁺ is added (Matsudaet al., Biochem, and Mol. Biol. International 30, 419-424, 1993). InTable 2 it can be seen that the recombinant Mts1 protein at aconcentration 1.5 mg/ml had a broad spectrum of molecular weightsranging from 28.9 kDa for dimer, 47.2 kDa for tetramer, and up to143.0-200.0 kDa for polymeric molecules. TABLE 2 Dynamic LightScattering Parameters D_(t) R_(h) M.W (kDa) M.W (kDa) Oligomeric State(le-9*cm/s {circumflex over ( )}2) nm First Model Second Model Dimers785 2.56 28.9 nd Tetramers 636 3.16 47.2 nd Oligomers 398 4.99 143.0200.0

[0096]

1 2 1 101 PRT Homo sapiens 1 Met Ala Cys Pro Leu Glu Lys Ala Leu Asp ValMet Val Ser Thr Phe 1 5 10 15 His Lys Tyr Ser Gly Lys Glu Gly Asp LysPhe Lys Leu Asn Lys Ser 20 25 30 Glu Leu Lys Glu Leu Leu Thr Arg Glu LeuPro Ser Phe Leu Gly Lys 35 40 45 Arg Thr Asp Glu Ala Ala Phe Gln Lys LeuMet Ser Asn Leu Asp Ser 50 55 60 Asn Arg Asp Asn Glu Val Asp Phe Gln GluTyr Cys Val Phe Leu Ser 65 70 75 80 Cys Ile Ala Met Met Cys Asn Glu PhePhe Glu Gly Phe Pro Asp Lys 85 90 95 Gln Pro Arg Lys Lys 100 2 101 PRTMus musculus 2 Met Ala Arg Pro Leu Glu Glu Ala Leu Asp Val Ile Val SerThr Phe 1 5 10 15 His Lys Tyr Ser Gly Lys Glu Gly Asp Lys Phe Lys LeuAsn Lys Thr 20 25 30 Glu Leu Lys Glu Leu Leu Thr Arg Glu Leu Pro Ser PheLeu Gly Lys 35 40 45 Arg Thr Asp Glu Ala Ala Phe Gln Lys Val Met Ser AsnLeu Asp Ser 50 55 60 Asn Arg Asp Asn Glu Val Asp Phe Gln Glu Tyr Cys ValPhe Leu Ser 65 70 75 80 Cys Ile Ala Met Met Cys Asn Glu Phe Phe Glu GlyCys Pro Asp Lys 85 90 95 Glu Pro Arg Lys Lys 100

We claim:
 1. An isolated functional derivative of an Mts protein.
 2. Anisolated Mts1-del75.
 3. An isolated Mts1-4S.
 4. An isolated multimericMts1 protein complex, comprising at least three Mts1 protein molecules.5. The isolated multimeric Mts1 protein complex of claim 4 , having a MWin the range of about 30 kD to about 200 kD.
 6. The isolated multimericMts1 protein complex of claim 4 , wherein the Mts1 protein molecule iswild type.
 7. The isolated multimeric Mts1 protein complex of claim 4 ,wherein the Mts1 protein molecule is Mts1-del75.
 8. The isolatedmultimeric Mts1 protein complex of claim 4 , wherein the Mts1 proteinmolecule is of a mammalian origin.
 9. A pharmaceutical compositioncomprising the isolated functional derivative of an Mts1 protein ofclaim 1 , and a pharmaceutically acceptable carrier.
 10. Apharmaceutical composition comprising the isolated complex of claim 4 ,and a pharmaceutically acceptable carrier.
 11. The pharmaceuticalcomposition of claim 9 or 10 , wherein said pharmaceutically acceptablecarrier is liquid, semi-solid, or solid.
 12. The pharmaceuticalcomposition of claim 9 or 10 , further comprising a neurotropic factor.13. The pharmaceutical composition of claim 12 , wherein saidneurotropic factor is selected from the group consisting of bFGF, aFGF,CNTF, NGF, BDNF, GDNF, NT3, NT4/5, IGF-1 and IGF-II.
 14. A method ofstimulating growth of neuronal cells, comprising administering an Mts1protein or a functional derivative thereof to said neuronal cells.
 15. Amethod of treating a neurological condition in a subject, wherein saidneurological condition is characterized by neuronal degeneration, deathor injury, comprising administering to the subject a therapeuticallyeffective amount of an Mts1 protein or a functional derivative thereofand a pharmaceutically acceptable carrier.
 16. A method of treating aneurological condition in a subject, wherein said neurological conditionis characterized by neuronal degeneration, death or injury, comprisingadministering to the subject a therapeutically effective amount of anMts1 protein-encoding nucleic acid sequence and a pharmaceuticallyacceptable carrier.
 17. The method of claim 16 , wherein said nucleicacid sequence is provided in an expression vector.
 18. The method ofclaim 16 , wherein said expression vector is a plasmid, retroviral,adenoviral, herpes simplex viral, adeno-associated viral, polio viral ora vaccinia vector.
 19. The method of claims 15 or 16, wherein saidneurological condition is Parkinson's disease, Alzheimer's disease,Down's Syndrome, stroke, cardiac arrest, sciatic crush, spinal cordinjury, injury to sensory neurons, or degenerative disease of theretina.
 20. The method of claim 19 , further comprising administeringsimultaneously a neurotropic factor.
 21. The method of claim 20 ,wherein said neurotropic factor is selected from the group consisting ofbFGF, aFGF, CNTF, NGF, BDNF, GDNF, NT3, NT4/5, IGF-1 and IGF-II.
 22. Themethod of claim 19 , wherein the administration is via an oral,ophthalmic nasal, topical, transdermal, intravenous, intraperitoneal,intradermal, subcutaneous or intramuscular, intracranial, intracerebral,intraspinal, intravaginal, intrauterine, or rectal route.
 23. The methodof claim 19 , wherein the administration is via implantation.